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Neuronal and Glial Growth in Organotypic Cultures after Vitrification

Arav Amir, Shahar Abraham, Ziv-Polat Ofra, Natan Yehudit and Patrizio Pasquale

Fetal rat dorsal root ganglia (DRG) and spinal cords (SC) slices from rat fetuses were vitrified in a new semiautomatic vitrification system, cooled in sterile slush liquid air (SLA) and stored in a special sterile sealed container in liquid nitrogen (LN). Upon warming organotypic stationary cultures were performed in using NVR-Gel (composed mainly from hyaluronic acid and laminin) and enriched with neuronal factors conjugated to iron oxide nanoparticles. Evaluation of cultures was made by daily phase-contrast microscopy observations and by immune fluorescent staining.
Results revealed that SC neurons maintained their multipolar shape and regrew dendrites and axons. The round shape DRG neurons exhibited euchromatic nuclei with prominent nucleoli and an active regeneration of nerve processes. Migration of both neurons and flat cells (fibroblasts and glia Schwann cells) started within 48 hours after seeding and intensified in the upcoming days.
In conclusion, it can be said that the using a semi-automatic vitrification, sterile vitrification and sterile storage of neuronal tissues from the CNS and the PNS is a successful advanced technology for the preservation of neurons and glial cells, as shown in the regain of a full regular growth pattern in culture. This may an important step towards clinical use in the reconstruction of severe peripheral nerves and spinal cord injuries.